Enhanced Endoglucanase Production by Bacillus aerius on ... · Enhanced Endoglucanase Production by Bacillus aerius on Mixed Lignocellulosic Substrates Mushafau Adebayo aOke,,b Mohamad

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Oke et al. (2016). “Endoglucanase production,” BioResources 11(3), 5854-5869. 5854

Enhanced Endoglucanase Production by Bacillus aerius on Mixed Lignocellulosic Substrates

Mushafau Adebayo Oke,a,b Mohamad Suffian Mohamad Annuar,a and

Khanom Simarani a,*

Selected carbon sources including soluble carboxymethyl cellulose (CMC), insoluble microcrystalline cellulose (MCC), and single (SS)/mixed lignocellulosic substrates (MS), were evaluated for endoglucanase production by B. aerius S5.2. The lignocellulosic substrates of oil palm empty fruit bunch (EFB), oil palm frond (OPF), rice husk (RH), and their mixture (MS) supported growth of the strain better than CMC and MCC. The maximum endoglucanase activity on MS was 7.3-, 2.6-, 1.7-, and 1.2-fold higher than those recorded on MCC, CMC, EFB/OPF, and RH, respectively. While the reducing sugar concentration of the CMC medium was comparable to that of the EFB and MS media, wide variability was observed in the reducing sugar concentrations among the lignocellulosic substrates. Extremely low levels of sugar were detected in the MCC medium, reflecting its poor digestibility and hence unsuitability for growth and endoglucanase production. Endoglucanase production was predominantly extracellular when the strain was grown on CMC and MS. After seven days of fermentation, there was an approximately 25% reduction in MS dry weight. These findings show that the use of mixed lignocellulosics could potentially reduce the cost of cellulase production. Certain novel aspects of the cellulase system of B. aerius are reported in this study.

Keywords: Cellulase; Endoglucanase; Mixed lignocellulosic substrate; Bacillus aerius; Bioprocessing

Contact information: a: Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603

Kuala Lumpur, Malaysia; b: Department of Microbiology, Faculty of Life Sciences, University of Ilorin,

P.M.B. 1515, Ilorin, Nigeria; *Corresponding author: [email protected]; [email protected]

INTRODUCTION

Cellulases, a group of synergistic enzymes that hydrolyze β-glycosidic bonds in

cellulose, are crucial in the valorization of lignocellulosic biomass into value-added

products. Cellulases are categorized into three major groups: endoglucanases,

exoglucanases, and β-glucosidases (Lynd et al. 2002). Although the concerted action of

these three cellulase types enhance cellulose hydrolysis, this feature may be undesirable in

some situations because of the varied activities and substrate specificities of the individual

cellulase components (Puranen et al. 2014). Hence, it may be necessary to focus on the

production of certain cellulase types that exhibit properties more suitable for specific

applications. Endoglucanases are of special interest because of their ability to initiate

cellulose hydrolysis, and their action on the amorphous regions of crystalline cellulose

matrix is considered the rate-limiting step of cellulose utilization (Malherbe and Cloete

2002). Furthermore, endoglucanases have special applications in textile and food

processing industries (Juturu and Wu 2014; Puranen et al. 2014).



The commercial production of cellulases has been carried out predominantly using

fungi because fungi produce higher titers than bacteria. Bacterial cellulases may be a

competitive commercial product for the following reasons: high growth rate of bacteria,

high stability, resistance to relatively harsh conditions, genetic amenability, and production

of efficient multi-enzyme complexes (cellulosomes) (Maki et al. 2009, 2011).

The quantity and quality of cellulase produced by microorganisms is strongly

dependent on the carbon source (Evans et al. 1992; Dashtban et al. 2011) and is controlled

by repression and induction mechanisms (Bisaria and Mishra 1989; James and Ming 1991).

The carbon source also influences the location of cellulases in the cell. Cellulases are

expressed differentially in various cell locations when grown on different substrates (Berg

1975; Lo et al. 2009).

Various carbon sources such as disaccharides (e.g., cellobiose, lactose, and

sophorose) and pure soluble/insoluble cellulosics (carboxymethyl cellulose -CMC, Avicel,

Solka floc, etc.) are good inducers of cellulase synthesis; however, the use of these

substrates on a commercial scale is uneconomical because of their high cost (Chen and

Wayman 1991). In fact, sensitivity analysis has shown that the carbon source is the major

cost-factor in cellulase production (Ryu and Mandels 1980). Hence, researchers have

explored the use of lignocellulosic biomass as the substrate for cellulase production

because it is inexpensive and abundant.

Studies on the use of lignocellulosics for bacterial cellulase production have

focused mainly on the use of single substrates (Assareh et al. 2012; Da Vinha et al. 2011;

Harun et al. 2013; Krishna 1999). However, this approach might be unsustainable in a real

biorefinery situation because the feedstock supply is subject to seasonal variation and other

logistical problems (Allen et al. 1998; Rentizelas et al. 2009; Sokhansanj and Hess 2009).

The use of mixed substrates in biorefineries can lower logistic costs as well as eliminate

the need for extraneous nutrient supplementation (Martín et al. 2008; Thomsen and

Haugaard-Nielsen 2008; Sultana and Kumar 2011). Consequently, the use of mixed

substrates is an interesting option for lowering bacterial cellulase production costs.

Some members of the genus Bacillus are good cellulase producers (Acharya and

Chaudhary 2012; Asha and Sakthivel 2014; Balasubramanian and Simões 2014; Gaur and

Tiwari 2015), while some Bacillus spp. produce cellulases that are stable under extreme

conditions (Rastogi et al. 2010; Annamalai et al. 2011; Trivedi et al. 2011). However,

cellulase production has not been reported previously for B. aerius. This species was first

classified in 2006, after Shivaji et al. (2006) isolated the bacterium from cryogenic tubes

used for sampling air at high altitudes. The most commonly reported enzyme of

biotechnological interest is lipase (Saun et al. 2014a; Saun et al. 2014b; Narwal et al.

2015), although Oliveira et al. (2016) recently reported the isolation of a protease-

producing strain from waste feathers.

B. aerius S5.2 used in this study was isolated from decomposing oil palm empty

fruit bunch (EFB) samples in Malaysia. This strain showed the unique ability to produce

endoglucanase from mixed rice and oil palm residues (unpublished). Thus, the strain was

selected as a model organism to study bacterial endoglucanase production from mixed

lignocellulosic substrates. Furthermore, little is known about the cellulolytic system of B.

aerius, and this study investigated the effects of various single and mixed carbon sources

on endoglucanase production, as well as the location(s) of endoglucanase, when the

bacterium was grown on different substrates. The utilization of the mixed substrate by B.

aerius was determined from the extent of degradation after seven days of cultivation.



EXPERIMENTAL

Bacterial Strain Bacillus aerius S5.2 was isolated from decomposing EFB residues collected from

an oil palm plantation in Kuala Selangor, Malaysia. This strain produced high titers of

endoglucanase on a mixed substrate (MS) medium comprised of EFB, oil palm frond

(OPF), and rice husk (RH). The bacterium was identified as B. aerius following sequencing

of the 16S rRNA gene and a sequence similarity check using the BLAST tool on the NCBI

database (http://www.ncbi.nlm.nih.gov). The sequence was submitted to GenBank, and the

accession number (KP178216) was obtained. The cell morphology was observed using

field emission scanning electron microscopy (FESEM) (JSM-7001F, JOEL, Tokyo,

Japan). Gram staining and the non-staining KOH methods (Buck 1982) were used to

determine the Gram reaction of the strain. Stock cultures of the strain were stored on

nutrient agar slants and maintained at 4 °C with regular sub-culturing. This strain was

deposited at the Microbial Culture Collection Unit (UNiCC) of the Institute of Bioscience,

Universiti Putra Malaysia, under accession number UPMC 1179.

Carbon Sources for Endoglucanase Production The carbon sources used for the induction of endoglucanase production are

presented in Table 1. Medium without cellulosic carbon sources was used as the control in

the experiments.

Table 1. Carbon Sources for Endoglucanase Production by B. aerius S5.2

Pure Cellulosic Substrate Lignocellulosic Substrate

CMC -

MCC EFB, OPF, RH, MS

CMC: soluble carboxymethyl cellulose; MCC: insoluble microcrystalline cellulose; EFB: oil palm empty fruit bunch; OPF; oil palm frond; RH: rice husk; MS: mixed substrate.

Fresh OPF samples were obtained from the Malaysian Palm Oil Board (MPOB),

Bangi, Malaysia. The leaflets were removed, and the fresh petioles were cut into smaller

pieces and dried under sunlight. Only the petioles were used in this study because OPF

leaflets have other important uses in an oil palm plantation. The petiole has high sugar

levels, which makes it more desirable for use as a feedstock in biofuels and other

bioproducts (Zahari et al. 2012).

Dried and shredded EFB fibers were obtained from the Biocomposting Pilot

Facility, Universiti Putra Malaysia. Rice husks were collected from a paddy field in Kedah,

Malaysia. The three biomass samples were reduced to small particles (300 to 425 µm)

using a Rapid granulator (GK 205-K, Terramar GmbH, Hamburg, Germany). The MS

carbon source was prepared by mixing EFB, OPF, and RH in equal proportions (1:1:1).

The mixture was stored in a dry airtight container.

The lignocellulosic substrates used in this study were pretreated sequentially with

NaOH and autoclaved, according to the method in Umikalsom et al. (1997), with a slight

extension of the autoclaving duration to 15 min. The washed and pretreated solids were

dried in an oven at 60 °C for at least 12 h.



Culture Media A modified version of the medium for the cultivation of cellulolytic bacteria,

described by Dickerman and Starr (1951) and containing 2% (w/v) of the respective carbon

sources, was used for endoglucanase production. Other components of the medium were

(w/v): K2HPO4 (0.08%), KH2PO4 (0.02%), MgSO4·7H2O (0.02%), NaCl (0.02%), NaNO3

(0.1%), CaCO3 (0.001%), and yeast extract (0.05%). The medium pH was adjusted to 7.0

using 2 M NaOH or HCl. The media was sterilized at 121 °C for 15 min in an autoclave.

Growth and Endoglucanase Production B. aerius S5.2 was cultivated in nutrient broth until the late log phase (12 h) was

reached. Aliquots (approximately 107 cfu/mL) from this culture were used as inocula in the

experiments. Five percent inoculum (v/v) was inoculated into 250-mL Erlenmeyer flasks

containing the culture media with the respective carbon source. Each flask was incubated

at 30 °C with 200 rpm agitation for 72 h. Triplicate flasks were used for each carbon source.

Aliquots of the culture samples were initially collected after 6 h and then at 12-h intervals.

Collected samples were centrifuged at 6000 rpm for 10 min at 4 C. The cell-free

supernatant was used as the crude enzyme preparation in the endoglucanase assay. Growth

of the bacterium at each sampling period was monitored by estimating the total colony-

forming units (cfu) in the culture supernatant using the drop plate technique (Herigstad et

al. 2001).

Reducing Sugar Production In order to evaluate the general digestibility and suitability of each of the substrates

for growth and utilization by B. aerius S5.2, the amount of reducing sugar in the culture

supernatant at each sampling period was monitored. Reducing sugar concentrations were

determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959).

Localization of Endoglucanase To determine the localization of endoglucanase in the cell, B. aerius S5.2 was

grown in the culture media with 2% (w/v) of either CMC or pre-treated MS as the carbon

source at 30 °C for 36 h with 200 rpm agitation. The culture broth (30 mL) was centrifuged

at 8000 rpm for 10 min at 4 °C, and the supernatant was used as the extracellular enzyme

sample. The cell pellet was washed twice with 10 mL of 0.05 M phosphate buffer (pH 7.0)

and later re-suspended in 15 mL of the same buffer. The suspension was kept on ice to

preserve enzyme activity. The intracellular and membrane-bound fractions of the enzyme

were prepared by sonication (Lo et al. 2009). The cell pellet suspension was sonicated at

varying 30% amplitude using a probe type sonicator (Branson Ultrasonics, Danbury, CT,

USA). The sample was placed on ice and pulse-sonicated for 10 min for 30/10 sec pulse

intervals. This was followed by centrifugation at 8000 rpm for 10 min at 4 °C. The

supernatant was used as the intracellular enzyme sample. The cell pellet was resuspended

in 5 mL of buffer (to concentrate the sample) and used as the membrane-bound enzyme

sample. All enzyme fractions were analysed for endoglucanase activity and protein

concentration. Enzyme activity in the various fractions was expressed as enzyme units per

µg protein to account for the different volumes of buffer in the re-suspensions.



Protein Concentration Measurement Protein concentration was determined using the Bradford assay (Bradford 1976).

The enzyme suspension (100 µL) was mixed with 5 mL of Bradford reagent (Sigma-

Aldrich, St. Louis, MO, USA). The mixture was maintained at room temperature for 10

min, and the absorbance was read against the reagent blank (100 µL buffer plus 5 mL

reagent) at 595 nm.

The protein concentration was determined by extrapolation from a standard

calibration, which was computed using different concentrations (µg/mL) of bovine serum

albumin suspended in 0.05 M phosphate buffer vs. absorbance at 595 nm.

Endoglucanase Assay The endoglucanase activity was determined by measuring the reducing sugars

released after 200 µL of the enzyme was reacted with 200 µL of 2% CMC in 0.05 M

phosphate buffer at pH 7.0 (Zhang et al. 2009). The mixture was incubated for 30 min at

50 °C, and the reaction was stopped by adding 800 µL of DNS reagent, followed by

immersion in boiling water for 5 min. The released sugars were measured as glucose

equivalents using the DNS reagent (Miller 1959). One unit (U) of enzyme activity was

defined as the amount of enzyme that liberated 1 µmol of reducing sugar per mL per minute

from the substrate.

Degradation of MS by B. aerius S5.2 Five millilitres of B. aerius S5.2 inoculum from the late log phase was transferred

into a 250-mL conical flask containing 50 mL of medium with 1 g of MS as the carbon

source. The flask was incubated for seven days at 30 C with 200 rpm agitation. A control

was prepared in a separate flask containing the same amount of substrate without inoculum.

All experiments were conducted in triplicate. The extent of the degradation of MS was

determined by calculating the dry weight loss of the substrate. After the cultivation period,

the entire content of each flask was filtered through dry Whatman No. 1 filter paper (initial

weight; W0).

The liquid was allowed to drain completely, and the residue with the filter paper

was dried in an oven at 70 C until a constant weight (W1) was obtained. The extent of the

degradation of MS was expressed as the percentage of dry weight loss of the substrate as

follows:

1.0 − (𝑊1 − 𝑊0) × 100% (1)

The numerical value 1.0 represents the initial amount of MS (1 g) for the

degradation experiment. The mean of three replicates was recorded as the final dry weight

loss.

RESULTS AND DISCUSSION

Morphology of B. aerius S5.2 The morphology of B. aerius S5.2 was observed using FESEM. The Gram reaction

was also confirmed using normal Gram staining as well as the non-staining KOH method.

B. aerius S5.2 cells were Gram-positive and rod-shaped. Micrograph images of the cell

morphology are presented in Fig. 1. The cell size was 0.40 to 0.50 µm by 1.22 to 2.49 µm,

while the endospores ranged from 0.23 to 0.26 µm in size.



Fig. 1. Field emission scanning electron microscopy imaging of B. aerius S5.2. (a) Cells ( 5,000);

(b) cells and endospores ( 20,000)

Growth and Endoglucanase Production on Various Cellulosic Carbon Sources

The growth curve and endoglucanase production profile of B. aerius S5.2 during

growth on various cellulosic carbon sources are presented in Figs. 2 and 3, respectively.

The bacterium showed better growth on the lignocellulosic substrates (single and mixed)

than on pure cellulosic substrates (CMC and MCC). The least suitable substrate for growth

was MCC (9.07 log cfu), followed by CMC (11.52 log cfu), because the maximum growth

rates were inferior. The highest growth was recorded on EFB (max. 16.38 log cfu) and OPF



(max. 15.46 log cfu). The maximum growth value recorded on MS (12.96 log cfu) was

comparable to that recorded on RH (12.92 log cfu), although the growth rate of B. aerius

S5.2 was higher on the latter.

A similar trend was also observed for endoglucanase production (Fig. 3). The MCC

and CMC produced the lowest enzyme titers, while higher titers were recorded for

lignocellulosic substrates. The maximum endoglucanase activity recorded on MCC was

0.108 0.050 U/mL, while on CMC, it was 0.305 0.063 U/mL. However, the maximum

endoglucanase titers recorded on MS (0.787 0.062 U/mL) and RH (0.658 0.019 U/mL)

were the highest among all of the carbon sources investigated. Endoglucanase production

on EFB and OPF was similar, with maximum titers of 0.470 0.056 U/mL and 0.463

0.007 U/mL, respectively. Based on the maximum enzyme titers obtained on each

substrate, it was observed that the MS supported endoglucanase production 7.3-, 2.6-, and

1.2- times better than MCC, CMC, and RH respectively. Also, MS supported 1.7-fold

higher endoglucanase titers compared with OPF and EFB.

Figures 2 and 3 show that endoglucanase production was growth-related as the

strain’s growth and endoglucanase production showed similar profiles. Similar observation

was previously reported for Bacillus spp. (Ariffin et al. 2006). The higher cell growth and

endoglucanase production recorded on the lignocellulosic substrates could be attributed to

the availability of more growth-promoting substances. Lignocellulosic biomass materials

have various proteins in addition to cellulose, hemicellulose, lignin, other components

(Sluiter et al. 2008). These substances may improve the metabolism of the bacterium in

comparison to the pure cellulosic carbon sources. Yang et al. (2014) compared CMCase

production on CMC, MCC, rice hull, and wheat bran by B. subtilis BY-2. The authors

reported that the highest enzyme production was obtained with wheat bran, while rice hull

and CMC yielded lower enzyme values. A very low CMCase titer was recorded on MCC.

Similar observations were reported by Chan and Au (1987), where B. subtilis AU-1

produced lower cell yields and CMCase when grown on pure cellulosics, such as Sigmacell

20 and filter paper, compared with other carbon sources. In contrast, Harun et al. (2013)

reported that MCC supported higher cellulase production by Thermobifida fusca than

pretreated EFB. However, this observation could be attributed to strain differences and the

fact that the authors dried their substrates at 105 °C after the pretreatment, while the

substrates used in this study were dried at 60 °C. The high temperature employed for drying

the EFB may have destroyed protein components of the substrate, thereby reducing the

nutrients available for growth and enzyme production.

One interesting observation from these results is that the maximum endoglucanase

titer of MS was significantly higher (P < 0.05) than the pure cellulosics, or with EFB and

OPF. The only exception was RH, which showed comparable titers with MS. This result

was possibly due to the combination of favourable characteristics (e.g., nutrients, cellulose

accessibility, etc.) for each individual lignocellulosic material in the mixture. The strategy

of using mixed substrates could reduce the cost of cellulase production, which is currently

based on expensive synthetic substrates. Furthermore, the use of mixed lignocellulosics

facilitates the management of feedstock supply fluctuations (Nilsson and Hansson 2001).

This approach also reduces delivery costs to the biorefinery compared with the use of single

type feedstocks (Sultana and Kumar 2011). Studies on other applications of lignocellulosic

mixtures have demonstrated that combining substrates usually has no negative impacts on

product yields; more often than not, higher yields were obtained on mixtures than on the

single substrates. Such observations have been reported with respect to the pretreatment



and hydrolysis of mixed substrates (Jensen et al. 2008; Moutta et al. 2013; Moutta et al.

2014; Pereira et al. 2015), bioethanol production (Erdei et al. 2010; Pereira et al. 2015),

and fungal cellulase production (Olsson et al. 2003).

Fig. 2. Growth curve of B. aerius S5.2 on various pure cellulosic and lignocellulosic substrates. Error bars represent standard deviations.

Fig. 3. Endoglucanase production by B. aerius S5.2 on various pure cellulosic and lignocellulosic substrates. Error bars represent standard deviations.

Reducing Sugar Production The reducing sugar profile of the strain was monitored in the culture supernatants

during growth on the carbon sources (Fig. 4). The amount of reducing sugar released into

the medium was an indication of the relative digestibility of a substrate, and it also was an



adequate indication of the bacterial cellulolytic ability (Han and Callihan 1974). Hence,

reducing sugars in the culture medium were generated as a result of the hydrolytic activity

of the enzymes secreted by the bacterium. The maximum amounts of reducing sugars

detected in the medium of each of the substrates during the entire fermentation period were

compared. These were generally higher than the initial reducing sugar concentrations (0.00

to 0.01 mg/mL) in the media prior to fermentation. Extremely low levels of reducing sugar

were detected in the MCC medium because it was a poor carbon source for growth and

enzyme production (Figs. 2 and 3). Following MCC, the OPF medium had the lowest

concentration of sugar (0.07 mg/mL). Interestingly, the highest amount of sugar was

detected in the CMC medium (0.61 mg/mL), although this was not significantly higher (P

> 0.05) than the highest amount detected in the EFB (0.56 ± 0.10 mg/mL) and MS (0.42 ±

0.05 mg/mL) media. The maximum reducing sugar concentration of the RH medium (0.33

± 0.08 mg/mL) was comparable to that of the MS medium. This value was significantly

lower (P < 0.05) than those of CMC and EFB, but significantly higher (P < 0.05) than OPF.

A relatively high amount of reducing sugar was detected in the CMC medium,

despite the lower endoglucanase production. This result was attributed to the higher level

of hydrolysis of CMC compared with the other substrates. Endoglucanase has high

substrate specificity for CMC and lower specificity for crystalline forms of cellulose (Kim

1995; Dobrev and Zhekova 2012; Miotto et al. 2014). Hence, endoglucanase, produced by

the medium, must have hydrolysed the CMC better, thus producing higher amounts of

reducing sugar than the other substrates. The differences in the levels of reducing sugars

produced by the media of the lignocellulosic substrates are probably related to differences

in the structural and physicochemical characteristics of the substrates, as a result of the

pretreatment. Pretreatment causes changes in the properties (e.g., chemical composition,

crystallinity, accessible surface area, porosity, etc.) of lignocellulosic substrates, which

consequently results in varying degrees of digestibility with cellulase (Meng and

Ragauskas 2014). Although the chemical composition of the lignocellulosic substrates

used in this study was not determined, it is possible that the pretreatment caused the

retention of higher amounts of lignin by the OPF, which may have resulted in the low

digestibility on contact with endoglucanase. Lignin exerts an inhibitory effect on cellulases

(Rahikainen et al. 2013; Gao et al. 2014).

Fig. 4. Reducing sugar profile of the culture supernatants of B. aerius S5.2 during its growth on pure cellulosic and lignocellulosic substrates. Error bars represent standard deviations.



Localization of Endoglucanase on CMC and MS The localization of the endoglucanase activities of B aerius S5.2 grown on a soluble

pure cellulosic carbon source and a lignocellulosic substrate was investigated. CMC was

chosen as the pure cellulosic source because it resulted in higher endoglucanase activity

than the MCC. Likewise, MS was chosen as the lignocellulosic substrate because it

supported the highest endoglucanase production. This was done to investigate the

expression pattern of the enzyme in relation to the cellular location when the bacterial strain

was grown on different substrates (Fig. 5).

Endoglucanase production was predominantly extracellular, irrespective of the

substrate solubility. As observed in the earlier carbon source experiments, endoglucanase

production on MS was significantly higher (P < 0.05) than on CMC extracellularly. These

observations were in agreement with results by Kricke et al. (1994), who found that

CMCase was produced both extracellularly and intracellularly by a Bacillus spp. isolated

from termite mount soils. The extracellular production of enzymes among Bacillus spp. is

common (Priest 1977; Molva et al. 2009). Some endoglucanase activity was also detected

as intracellular and membrane-bound when B. aerius S5.2 was grown on both substrates;

however, this expression was minimal compared with extracellular production. Cell-bound

(intracellular and membrane-bound) cellulase activity in bacteria is believed to represent a

basal level of enzyme expression, reflecting the synthesis of cellulase by induction,

transcription, and translation within the cell. Hence, cellulases are initially cell-bound

before secretion into the medium (Gong and Tsao 1979). The higher extracellular

expression of endoglucanase on MS and CMC is a reflection of the nature of both substrates

because they both contain amorphous regions for which endoglucanase has high

specificity. Furthermore, cellulases that are required for the hydrolysis of a particular

substrate are usually expressed extracellularly (Ramasamy and Verachtert 1980).

Fig. 5. Cellular location of B. aerius S5.2 endoglucanase during its growth on MS and CMC. Error bars represent standard deviations. Within the same location, bars that share the same letters are not significantly different (P > 0.05).

The extracellular production of enzymes by microorganisms has promising

implications because such enzymes are easier to purify and can be less prone to proteolytic



attack compared with intracellular enzymes (Gao et al. 2015). Furthermore, extracellular

secretion of cellulase by any strain is useful in developing consolidated bioprocessing

(CBP) systems where cellulase production, cellulose hydrolysis, and fermentation occur

simultaneously (Lynd et al. 2002; Gao et al. 2015). Because extracellular cellulase is

available for the liberation of fermentable sugars, they could be immediately utilized by

the fermenting microorganism.

Degradation of MS Following the higher expression of endoglucanase by B. aerius S5.2 on MS, the

ability to utilize MS was later assessed by substrate gravimetric dry weight loss after some

period of cultivation. The gravimetric method evaluates the cellulolytic ability of bacteria

on different lignocellulosic substrates (Gupta et al. 2012; Maki et al. 2014).

A total of 25.3 ± 2.5% of substrate dry weight loss occurred with MS compared

with the control flask (19% dry weight loss). This difference was statistically significant

(P = 0.049). The weight loss recorded from the control was primarily attributed to the

dissolution of part of the substrate into the medium due to continuous agitation. Table 2

shows that the culture attained high cell growth on the MS after 72 h, with a cell yield

reaching 12.96 log cfu/mL. However, at the end of the 7 d cultivation, the growth had

declined to 9.88 log cfu/mL. It is likely that the bacterium was supported almost entirely

by the soluble compounds of the MS during the period of active growth, thus resulting in

the slight difference between the experimental and the control samples. Thus, it appears

that the nature of lignocellulosic biomass affected the utility of the gravimetric method to

assess cellulolytic ability of bacterial culture on certain substrates.

CONCLUSIONS

1. Cheap and abundant mixed lignocellulosics are promising for the commercial

production of endoglucanase, where the cost of production might be reduced.

2. Higher amounts of extracellular endoglucanase were produced on mixed

lignocellulosics by B. aerius S5.2 and could be advantageous in terms of enzyme

recovery and use in CBP.

3. This study also provided insights into the less explored aspects of B. aerius cellulolytic

system.

4. The B. aerius strain is promising and worth investigating further for possible

application in the commercial production of endoglucanase production from

lignocellulosics.

ACKNOWLEDGMENTS

This research was funded by the University of Malaya with the research grants

RP024-2012D, RG048-11BIO, and PG114-2013B.



REFERENCES CITED

Acharya, S., and Chaudhary, A. (2012). "Optimization of fermentation conditions for

cellulases production by Bacillus licheniformis MVS1 and Bacillus sp. MVS3

isolated from Indian hot spring," Brazilian Archives of Biology and Technology 55,

497-503.

Allen, J., Browne, M., Hunter, A., Boyd, J., and Palmer, H. (1998). "Logistics

management and costs of biomass fuel supply," International Journal of Physical

Distribution & Logistics Management 28(6), 463 - 477.

Annamalai, N., Thavasi, R., Vijayalakshmi, S., and Balasubramanian, T. (2011). "A

novel thermostable and halostable carboxymethylcellulase from marine bacterium

Bacillus licheniformis AU01," World Journal of Microbiology and Biotechnology

27(9), 2111-2115.

Ariffin, H., Abdullah, N., Umi Kalsom, M., Shirai, Y., and Hassan, M. (2006).

"Production and characterization of cellulase by Bacillus pumilus EB3," International

Journal of Engineering and Technology 3(1), 47-53.

Asha, B., and Sakthivel, N. (2014). "Production, purification and characterization of a

new cellulase from Bacillus subtilis that exhibit halophilic, alkalophilic and solvent-

tolerant properties," Annals of Microbiology 64(4), 1839-1848.

Assareh, R., Shahbani Zahiri, H., Akbari Noghabi, K., Aminzadeh, S., and Bakhshi

Khaniki, G. (2012). "Characterization of the newly isolated Geobacillus sp. T1, the

efficient cellulase-producer on untreated barley and wheat straws," Bioresource

Technology 120, 99-105.

Balasubramanian, N., and Simões, N. (2014). "Bacillus pumilus S124A carboxymethyl

cellulase; A thermo stable enzyme with a wide substrate spectrum utility,"

International Journal of Biological Macromolecules 67, 132-139.

Berg, B. ( 1975). "Cellulase location in Cellvibrio fulvus," Canadian Journal of

Microbiology 21(1), 51-57.

Bisaria, V. S., and Mishra, S. (1989). "Regulatory aspects of cellulase biosynthesis and

secretion," Critical Reviews in Biotechnology 9(2), 61-103.

Bradford, M. M. (1976). "A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding," Analytical

Biochemistry 72(1), 248-254.

Buck, J. D. (1982). "Nonstaining (KOH) method for determination of gram reactions of

marine bacteria," Applied and Environmental Microbiology 44(4), 992-993.

Chan, K. Y., and Au, K. S. (1987). "Studies on cellulase production by a Bacillus

subtilis," Antonie Van Leeuwenhoek 53(2), 125-36.

Chen, S., and Wayman, M. (1991). "Cellulase production induced by carbon sources

derived from waste newspaper," Process Biochemistry 26(2), 93-100.

Da Vinha, F., Gravina-Oliveira, M., Franco, M., Macrae, A., da Silva Bon, E.,

Nascimento, R., and Coelho, R. (2011). "Cellulase production by Streptomyces

viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate," Applied

Biochemistry and Biotechnology 164(3), 256-267.

Dashtban, M., Buchkowski, R., and Qin, W. (2011). "Effect of different carbon sources

on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains,"

International Journal of Biochemistry and Molecular Biology 2(3), 274-286.



Dickerman, J., and Starr, T. (1951). "A medium for the isolation of pure cultures of

cellulolytic bacteria," Journal of Bacteriology 62(1), 133-134.

Dobrev, G. T., and Zhekova, B. Y. (2012). "Biosynthesis, purification and

characterization of endoglucanase from a xylanase producing strain Aspergillus niger

B03," Brazilian Journal of Microbiology 43, 70-77.

Erdei, B., Barta, Z., Sipos, B., Réczey, K., Galbe, M., and Zacchi, G. (2010). "Ethanol

production from mixtures of wheat straw and wheat meal," Biotechnology for

Biofuels 3(1), 1-9.

Evans, E. T., Wales, D. S., Bratt, R. P., and Sagar, B. F. (1992). "Investigation of an

endoglucanase essential for the action of the cellulase system of Trichoderma reesei

on crystalline cellulose," Journal of General Microbiology 138, 1639-1646.

Gao, D., Haarmeyer, C., Balan, V., Whitehead, T. A., Dale, B. E., and Chundawat, S. P.

(2014). "Lignin triggers irreversible cellulase loss during pretreated lignocellulosic

biomass saccharification," Biotechnology for Biofuels 7(1), 175.

Gao, D., Wang, S., Li, H., Yu, H., and Qi, Q. (2015). "Identification of a heterologous

cellulase and its N-terminus that can guide recombinant proteins out of Escherichia

coli," Microbial Cell Factories 14(1), 1-8.

Gaur, R., and Tiwari, S. (2015). "Isolation, production, purification and characterization

of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis

RG-07," BMC Biotechnology 15(1), 1-12.

Gong, C. S., and Tsao, G. T. (1979). "Cellulase biosynthesis and regulation," Annual

Reports on Fermentation Processes 3(11), 1-140.

Gupta, P., Samant, K., and Sahu, A. (2012). "Isolation of cellulose-degrading bacteria

and determination of their cellulolytic potential," International Journal of

Microbiology 2012, article ID 578925, 1-5.

Han, Y. W., and Callihan, C. D. (1974). "Cellulose fermentation: Effect of substrate

pretreatment on microbial growth," Applied Microbiology 27(1), 159-165.

Harun, N. A. F., Baharuddin, A. S., Zainudin, M. H. M., Bahrin, E. K., Naim, M. N., and

Zakaria, R. (2013). "Cellulase production from treated oil palm empty fruit bunch

degradation by locally isolated Thermobifida fusca," BioResources 8(1), 676-687.

Herigstad, B., Hamilton, M., and Heersink, J. (2001). "How to optimize the drop plate

method for enumerating bacteria," Journal of Microbiological Methods 44(2), 121-

129.

James, C. L., and Ming, S. (1991). "Bacterial cellulases: Regulation of synthesis," in:

Enzymes in Biomass Conversion, G. F. Leatham and M. E. Himmel (eds.), American

Chemical Society, Washington, DC, pp. 331-348.

Jensen, J., Morinelly, J., Aglan, A., Mix, A., and Shonnard, D. R. (2008). "Kinetic

characterization of biomass dilute sulfuric acid hydrolysis: Mixtures of hardwoods,

softwood, and switchgrass," AIChE Journal 54(6), 1637-1645.

Juturu, V., and Wu, J. C. (2014). "Microbial cellulases: Engineering, production and

applications," Renewable and Sustainable Energy Reviews 33, 188-203.

Kim, C. H. (1995). "Characterization and substrate specificity of an endo-beta-1,4-D-

glucanase I (Avicelase I) from an extracellular multienzyme complex of Bacillus

circulans," Applied and Environmental Microbiology 61(3), 959-965.

Kricke, J., Ouml, Rn, Varma, A., Miller, D., and Mayer, F. (1994). "Carboxymethyl

cellulase from Bacillus sp.: Isolation, macromolecular organization, and cellular

location," The Journal of General and Applied Microbiology 40(1), 53-62.



Krishna, C. (1999). "Production of bacterial cellulases by solid state bioprocessing of

banana wastes," Bioresource Technology 69(3), 231-239.

Lo, Y.-C., Saratale, G. D., Chen, W.-M., Bai, M.-D., and Chang, J.-S. (2009). "Isolation

of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for

cellulosic biohydrogen production," Enzyme and Microbial Technology 44(6–7), 417-

425.

Lynd, L. R., Weimer, P. J., Zyl, W. H. V., and Pretorius, I. S. (2002). "Microbial

cellulose utilization: Fundamentals and biotechnology," Microbiology and Molecular

Biology Reviews 66(3), 506–577.

Maki, M., Iskhakova, S., Zhang, T., and Qin, W. (2014). "Bacterial consortia constructed

for the decomposition of Agave biomass," Bioengineered 5(3), 165-172.

Maki, M., Leung, K. T., and Qin, W. (2009). "The prospects of cellulase-producing

bacteria for the bioconversion of lignocellulosic biomass," International Journal of

Biological Sciences 5(5), 500-516.

Maki, M. L., Broere, M., Leung, K. T., and Qin, W. (2011). "Characterization of some

efficient cellulase producing bacteria isolated from paper mill sludges and organic

fertilizers," International Journal of Biochemistry and Molecular Biology 2(2), 146-

154.

Malherbe, S., and Cloete, T. E. (2002). "Lignocellulose biodegradation: Fundamentals

and applications," Reviews in Environmental Science and Biotechnology 1(2), 105-

114.

Martín, C., Thomsen, M. H., Hauggaard-Nielsen, H., and BelindaThomsen, A. (2008).

"Wet oxidation pretreatment, enzymatic hydrolysis and simultaneous saccharification

and fermentation of clover–ryegrass mixtures," Bioresource Technology 99(18),

8777-8782.

Meng, X., and Ragauskas, A. J. (2014). "Recent advances in understanding the role of

cellulose accessibility in enzymatic hydrolysis of lignocellulosic substrates," Current

Opinion in Biotechnology 27, 150-158.

Miller, G. L. (1959). "Use of dinitrosalicylic acid reagent for determination of reducing

sugar," Analytical Chemistry 31(3), 426-428.

Miotto, L. S., de Rezende, C. A., Bernardes, A., Serpa, V. I., Tsang, A., and Polikarpov,

I. (2014). "The characterization of the endoglucanase Cel12A from Gloeophyllum

trabeum reveals an enzyme highly active on beta-glucan," PLoS One 9(9), e108393.

Molva, C., Sudagidan, M., and Okuklu, B. (2009). "Extracellular enzyme production and

enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains

isolated from cheese in Turkey," Food Control 20(9), 829-834.

Moutta, R. D. O., Ferreira-Leitão, V. S., and Bon, E. P. D. S. (2014). "Enzymatic

hydrolysis of sugarcane bagasse and straw mixtures pretreated with diluted acid,"

Biocatalysis and Biotransformation 32(1), 93-100.

Moutta, R. D. O., R, Silva, M., Reis Corrales, R., Santos Cerullo, M., and Ferreira-Leitão,

V. (2013). "Comparative response and structural characterization of sugarcane

bagasse, straw and bagasse-straw 1:1 mixtures subjected to hydrothermal

pretreatment and enzymatic conversion," Journal of Microbial and Biochemical

Technology S12, 2.

Narwal, S. K., Saun, N. K., Dogra, P., Chauhan, G., and Gupta, R. (2015). "Production

and characterization of biodiesel using nonedible castor oil by immobilized lipase

from Bacillus aerius," BioMed Research International 2015, 6.



Nilsson, D., and Hansson, P.-A. (2001). "Influence of various machinery combinations,

fuel proportions and storage capacities on costs for co-handling of straw and reed

canary grass to district heating plants," Biomass and Bioenergy 20(4), 247-260.

Oliveira, C. T., Pellenz, L., Pereira, J. Q., Brandelli, A., and Daroit, D. J. (2016).

"Screening of bacteria for protease production and feather degradation," Waste and

Biomass Valorization, 1-7.

Olsson, L., Christensen, T. M. I. E., Hansen, K. P., and Palmqvist, E. A. (2003).

"Influence of the carbon source on production of cellulases, hemicellulases and

pectinases by Trichoderma reesei Rut C-30," Enzyme and Microbial Technology

33(5), 612-619.

Pereira, S. C., Maehara, L., Machado, C. M., and Farinas, C. S. (2015). "2G ethanol from

the whole sugarcane lignocellulosic biomass," Biotechnology for Biofuels 8, 44.

Priest, F. G. (1977). "Extracellular enzyme synthesis in the genus Bacillus,"

Bacteriological Reviews 41(3), 711-753.

Puranen, T., Valtakari, L., Alapuranen, M., Szakacs, G., Ojapalo, P., and Vehmaanpera,

J. (2014). "Fungal endoglucanases, their production and use," AB Enzymes OY,

United States, 73.

Rahikainen, J. L., Martin-Sampedro, R., Heikkinen, H., Rovio, S., Marjamaa, K.,

Tamminen, T., Rojas, O. J., and Kruus, K. (2013). "Inhibitory effect of lignin during

cellulose bioconversion: The effect of lignin chemistry on non-productive enzyme

adsorption," Bioresource Technology 133, 270-278.

Ramasamy, K., and Verachtert, H. (1980). "Localization of cellulase components in

Pseudomonas sp. isolated from activated sludge," Microbiology 117(1), 181-191.

Rastogi, G., Bhalla, A., Adhikari, A., Bischoff, K. M., Hughes, S. R., Christopher, L. P.,

and Sani, R. K. (2010). "Characterization of thermostable cellulases produced by

Bacillus and Geobacillus strains," Bioresource Technology 101(22), 8798-8806.

Rentizelas, A., Tatsiopoulos, I., and Tolis, A. (2009). "An optimization model for multi-

biomass tri-generation energy supply," Biomass and Bioenergy 33(2), 223-233.

Ryu, D. D. Y., and Mandels, M. (1980). "Cellulases: Biosynthesis and applications,"

Enzyme and Microbial Technology 2(2), 91-102.

Saun, N. K., Mehta, P., and Gupta, R. (2014a). "Purification and physicochemical

properties of lipase from thermophilic Bacillus aerius," Journal of Oleo Science

63(12), 1261-8.

Saun, N. K., Narwal, S. K., Dogra, P., Chauhan, G. S., and Gupta, R. (2014b).

"Comparative study of free and immobilized lipase from Bacillus aerius and its

application in synthesis of ethyl ferulate," Journal of Oleo Science 63(9), 911-9.

Shivaji, S., Chaturvedi, P., Suresh, K., Reddy, G. S., Dutt, C. B., Wainwright, M.,

Narlikar, J. V., and Bhargava, P. M. (2006). "Bacillus aerius sp. nov., Bacillus

aerophilus sp. nov., Bacillus stratosphericus sp. nov. and Bacillus altitudinis sp. nov.,

isolated from cryogenic tubes used for collecting air samples from high altitudes,"

International Journal of Systematic and Evolutionary Microbiology 56(7), 1465-

1473.

Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., and Crocker, D.

(2008). Determination of Structural Carbohydrates and Lignin in Biomass, National

Renewable Energy Laboratory, Colorado.

Sokhansanj, S., and Hess, J. R. (2009). "Biomass supply logistics and infrastructure," in:

Biofuels: Methods and Protocols, J. R. Mielenz (ed.), Humana Press, New York, pp.

1-25.



Sultana, A., and Kumar, A. (2011). "Optimal configuration and combination of multiple

lignocellulosic biomass feedstocks delivery to a biorefinery," Bioresource

Technology 102(21), 9947-9956.

Thomsen, M. H., and Haugaard-Nielsen, H. (2008). "Sustainable bioethanol production

combining biorefinery principles using combined raw materials from wheat

undersown with clover-grass," Journal of Industrial Microbiology & Biotechnology

35(5), 303-311.

Trivedi, N., Gupta, V., Kumar, M., Kumari, P., Reddy, C. R. K., and Jha, B. (2011). "An

alkali-halotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva

lactuca," Carbohydrate Polymers 83(2), 891-897.

Umikalsom, M. S., Ariff, A. B., Zulkifli, H. S., Tong, C. C., Hassan, M. A., and Karim,

M. I. A. (1997). "The treatment of oil palm empty fruit bunch fibre for subsequent use

as substrate for cellulase production by Chaetomium globosum Kunze," Bioresource

Technology 62(1–2), 1-9.

Yang, W., Meng, F., Peng, J., Han, P., Fang, F., Ma, L., and Cao, B. (2014). "Isolation

and identification of a cellulolytic bacterium from the Tibetan pig's intestine and

investigation of its cellulase production," Electronic Journal of Biotechnology 17(6),

262-267.

Zahari, M. A. K. M., Zakaria, M. R., Ariffin, H., Mokhtar, M. N., Salihon, J., Shirai, Y.,

and Hassan, M. A. (2012). "Renewable sugars from oil palm frond juice as an

alternative novel fermentation feedstock for value-added products," Bioresource

Technology 110, 566-571.

Zhang, Y. H. P., Hong, J., and Ye, X. (2009). "Cellulase assays," in: Biofuels, J. R.

Mielenz (ed.), Humana Press, pp. 213-231.

Article submitted: March 1, 2016; Peer review completed: April 23, 2016; Revised

version received: May 3, 2016; Accepted: May 8, 2016; Published: May 16, 2016.

DOI: 10.15376/biores.11.3.5854-5869

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